Anti-heparin peptides

ABSTRACT

The invention concerns a compound exhibiting an anti-heparin activity, of formula Z B m ! (AXA) x  B n ! (AXA) y  B o  (AXA) z  B p , the diagnostic reagents comprising it and the use of said compound in an in vitro diagnostic test of a medicine for anti-heparin activity.

[0001] The present invention relates to a compound exhibitinganti-heparin activity, of formulaZ-B_(m)-(AXA)_(x)-B_(n)-(AXA)_(y)-B_(o)-(AXA_(z)-B_(p), to thediagnostic reagents comprising it and to the use of said compound in anin vitro diagnostic test or for producing a medicinal product foranti-heparin purposes.

[0002] Unfractionated heparin (UFH) is a natural sulfated polysaccharideof the glycosaminoglycan family. It is extracted essentially from pig orbovine intestinal mucosa.

[0003] It is a product which is heterogeneous by virtue of thecomposition of its saccharide units; as a result, the ionic charge ofthe molecule is also heterogeneous: different degree of sulfation of theglucosamine sulfate units and number of carboxylic functions differentin the iduronic and glucuronic acid units [1, 2].

[0004] The essential physical characteristics of heparin are: itspolyanionicity and its high charge density. Specifically, eachdisaccharide carries 3 to 4 negative charges (sulfates andcarboxylates).

[0005] A particular arrangement of the various units of heparin formsthe pentasaccharide reproducibly throughout the entire length of thesequence. This pentasaccharide is known to be the antithrombin IIIrecognition site of heparin, generating between these two entities ahigh affinity and, consequently, a high anticoagulant activity [3].

[0006] The chains containing no pentasaccharide are virtually devoid ofanticoagulant activity (only ⅓ of the heparin molecule exhibits thisaffinity for ATIII) [4].

[0007] Unfractionated heparin (UFH) is a polydisperse macromolecule:commercial UFH-based preparations have a molecular weight of between 12000 and 18 000 Da.

[0008] These preparations are mainly used for the purpose of preventiveor curative action against thromboses.

[0009] It is possible to fractionate UFH by size (after chemicaltreatment), which produces the family of LMWHs (low molecular weightheparins) [5].

[0010] UFH has equivalent activity on factors IIa and Xa. On the otherhand, LMWHs maintain a high anti-Xa activity and an anti-IIa activitywhich decreases with the size of the LMWH molecule.

[0011] Since a high anti-IIa activity may have hemorrhagic side effects,the LMWHs can be used advantageously as anticoagulants in place of UFHs.

[0012] However, the use of UFH or LMWH heparins in therapy may requirethe administration of compounds which neutralize their anticoagulanteffect, in particular at the end of interventions on the circulationsystem, such as extracorporeal circulation for example. Moreover, incertain diagnostic tests in vitro, the presence of heparin in thesamples may lead to erroneous interpretations of the results. Ittherefore proves to be necessary to also have compounds withanti-heparin properties for diagnosis.

[0013] Protamine sulfate is the only known compound capable ofneutralizing the anticoagulant effects of heparin in vivo. Protaminebelongs to the family of arginine residue-rich basic proteins, ispurified from salmon sperm [6-7] and neutralizes heparin by virtue ofits positive charges [8-10].

[0014] However, protamine causes hemodynamic and hematologic sideeffects such as hypotension, bradycardia, thrombocytopenia andleukopenia [11-15]. Specifically, it has been demonstrated that thepositive charges on protamine are directly proportional to the efficacyand neutralization of heparin, but also to the toxicity of the molecule[16].

[0015] Poly (L)-lysine and polybrene are other compounds known toneutralize heparin. However, these polycations also appear to be tootoxic for clinical use.

[0016] Wakefield et al. (WO 95/13083) describes protamine variants whichare thought to be less toxic. They are peptides having a 20 to 40 aminoacid sequence, with a positive charge of between +14 and +18, and thestructure of which corresponds to clusters of positive residuesinterspersed with neutral amino acids. Harris et al. (EP 999219)describes linear or branched anti-heparin peptides comprising asuccession of arginine and alanine clusters.

[0017] However, the abovementioned documents do not comprise anyteachings regarding the need to have anti-heparin agents in diagnosis.

[0018] A subject of the present invention is novel synthetic moleculeswhich are of use as heparin-neutralizing agents.

[0019] The properties of the molecules of the present invention havebeen more particularly studied in reagents of in vitro diagnosis.Besides their ability to be insensitive to the effects of the heparinpossibly present in the blood samples tested, it may also be necessaryfor some of these diagnostic reagents to exhibit low turbidity. Thisproperty is particularly important in the case of reagents containingthromboplastin for determining the Quick time, in particular onautomated devices with optical detection.

[0020] The molecules having general formula I explained below make itpossible to satisfy these two criteria. They therefore prove to be ofparticular use in neutralizing the possible interference associated withthe presence of heparin, both for diagnostic and for therapeuticpurposes.

[0021] Thus, the present invention relates to a compound exhibitinganti-heparin activity, of general formula I below:

Z-B_(m)-(AXA)_(x)-B_(n)-(AXA)_(y)-B_(o)-(AXA)_(z)-B_(p)

[0022] in which

[0023] *Z has the general formula:

[0024] with

[0025] Cy=-covalent bond

[0026] —CH₂—

[0027] —CO—

[0028] —CO—S—

[0029] —(CH₂)_(n)—X—

[0030] with 1≦n≦2 and X=O—, S—,

[0031] with

[0032] Cx=-covalent bond

[0033] —(CH₂)_(n)—, 1≦n≦5

[0034] or

[0035] with R1=—OH, —NH₂

[0036] *B represents a basic amino acid and can advantageously beselected from lysine, arginine, ornithine, histidine, homolysine andhomoarginine;

[0037] m, n, o and p represent, independently of one another, an integerof between 2 and 4.

[0038] Among the preferred compounds of the invention, the compounds inwhich m, n and o are equal to 4 and p is equal to 2 are moreparticularly noted.

[0039] *AXA represents a hydrocarbon-based chain corresponding to theformula:

[0040] —NH—(CH₂)_(n)—CO—, n being an integer of between 2 and 6;

[0041] x, y and z represent, independently of one another, an integer ofbetween 1 and 6.

[0042] Advantageously, Z is selected from the following compounds: Apc4-Aminopyrrolidine-2-carboxylic acid

Aze Azetidine-2-carboxylic acid

Cop 2-Carboxymorpholine acid

Disc 1,3-Dihydro-2H- isoindolecarboxylic acid

Hyp Hydroxyproline 4-Hydroxypyrrolidine-2- carboxylic acid

Inc Indoline-2-carboxylic acid

Inp Isonipecotic acid

PGlu Pyroglutamic acid 2-Pyrrolidone-5-carboxylic acid

Pip Pipecolic-2-carboxylic acid

Pro Proline Pyrrolidine-2-carboxylic acid

THC 2-Oxothiazolidine-4-carboxylic acid

Thi Thioproline Thiazolidinecarboxylic acid

Tiq Tetrahydroisoquinoline-2- carboxylic acid

[0043] Preferably, Z is pyroglutamic acid (PGlu).

[0044] Among the preferred compounds of the invention, the compounds inwhich AHA corresponds to NH—(CH₂)₅—CO—, i.e. aminohexanoic acid with xequal to 2, and y and z equal to 1, are more particularly noted.

[0045] Advantageously, the invention is directed toward the compoundcorresponding to the following formula:

PGlu-K₄-(AHA)₂-K₄-AHA-K₄-AHA-K₂

[0046] The compounds described above exhibit an anti-heparin capacity,determined by the Δ relative clotting time between a heparinized plasmaand a nonheparinized plasma, of less than 10%, preferably less than 5%.

[0047] A subject of the present invention is therefore also the use of acompound corresponding to formula 1 indicated above, in a reagent for invitro diagnosis, in particular reagents for calculating the clottingtime of a blood sample, or detecting or assaying various elements of theclotting cascade, independently of the presence or absence of heparin insaid sample.

[0048] Said reagents, in particular reagents of the PT (prothrombintime) type, advantageously exhibit a turbidity, measured by an OD at 630nm, of less than 0.5, preferably less than 0.1.

[0049] According to another aspect, the invention relates to a reagentfor in vitro diagnosis, comprising a compound as defined by generalformula 1 above, preferably a compound in which AXA corresponds toNH—(CH₂)₅—CO—, i.e. hexanoic amino acid, with x equal to 2, and y and zequal to 1, and more preferentially the compound below of formula:

PGlu-K₄-(AHA)₂-K₄-AHA-K₄-AHA-K₂

[0050] Such a diagnostic reagent thus makes it possible to carry outtests or assays in blood samples, independently of the presence orabsence of heparin in said samples.

[0051] A reagent according to the invention advantageously correspondsto conventional clotting tests, i.e. tests for determining the clottingtime of a blood sample, or to tests for detecting or assaying variouselements of the clotting cascade, such as, for example, assaying factorVIIa, fibrinogen, and proteins C and S, the PT test. These tests arecommonly used by those skilled in the art.

[0052] A preferred reagent for in vitro diagnosis according to theinvention is a reagent comprising thromboplastin, for carrying out a PT(prothrombin time or Quick time) test.

[0053] The final concentration in such reagents of the compoundaccording to the invention defined above is advantageously between 2 and10 μg/ml.

[0054] Preferably, this concentration is 5 μg/ml.

[0055] The invention also relates to the use of a compound of generalformula 1 defined above, as an anti-heparin agent for preparing amedicinal product.

EXAMPLE 1

[0056] Protocol for Synthesizing the Preferred Compound of theInvention:

[0057] The synoptic table of FIG. 1 summarizes the various steps of thesynthesis.

[0058] The structural chain is synthesized by solid-support chemistry,using a suitable automated device (Applied Biosystems 433A).

[0059] In order to make up the synthetic yield, the hydrophobic spacerarms (AXA) were added by double coupling.

[0060] The resin used is an MBHA (methyl benzyhydrylamine) resin, so asto recover a neutral amide C-terminal structure after cleavage from theresin.

[0061] The chemistry used here is Fmoc chemistry, using piperidine fordeprotection at each step.

[0062] The side chains are protected with Boc groups.

[0063] The scale of synthesis of the synthesizer is 0.1 nmole, producingapproximately 100 mg of crude product. The product is cleaved from theresin and deprotected on the side chains by treatment with TFA (55%trifluoroacetic acid). After concentration of the cleavage solution by ⅔of the volume, the product is precipitated from ether.

[0064] The crude product is subsequently lyophilized and then purifiedby reverse-phase HPLC, using a 5 μm Kromasil C18 column (20 mm×250 mm)with a gradient of water/acetonitrile 0.1% of TFA (from 0 to 60%acetonitrile in 30 min at 15 ml/minute).

[0065] Detection is carried out at 214 nm and the fractions arecollected manually.

[0066] The amount of purified product recovered is 50 mg.

[0067] The structure of the final product is confirmed by analysis ofthe sequence after hydrolysis with 6N HCl at 120° C. for 24 h; thehydrolyzate is then analyzed by reverse-phase HPLC (Kromasil C18 column,5 μm, 250 mm×4.6 mm), after derivatization with PITC(phenylthioisocyanate).

EXAMPLE 2

[0068] Test for Anti-Heparin Agents According to the Invention on a PTTest:

[0069] The aim is to analyze the performance (anti-heparin power andturbidity) of anti-heparin agents incorporated into a Diagnostica Stagothromboplastin reagent.

[0070] Analysis of the anti-heparin power: This is the ability of theanti-heparin agent to make the reagent insensitive to the heparincontained in the blood sample:

[0071] The test samples are prepared from a pool of normal plasma whichmay or may not be overloaded with heparin (plasma heparinized with 1IU/ml of UFH (Calciparine 25 000 IU/ml, Sanofi)).

[0072] Comment: a reagent serving as a negative control, i.e. withoutanti-heparin agent, is of use to verify the heparinization of theplasma.

[0073] To determine the Quick Time, the automated device (STADiagnostica Stago—patent EP 0325874) removes 50 μl of sample plasma,which it allows to incubate at 37° C. for 240 seconds in a cupulecontaining a bead; it then transfers the cupule into the measuring areaand adds 100 μl of thromboplastin reagent to the sample.

[0074] The clotting time was determined in the following way: Atconstant viscosity, the amplitude of oscillation of the bead in thecupule (oscillation maintained using an electromagnetic field) isconstant. When the viscosity increases (clotting phenomenon), theamplitude of oscillation of the bead decreases.

[0075] An algorithm uses this variation in amplitude to determine theclotting time.

[0076] The anti-heparin power is calculated in the following way:determination of the delta relative time between a heparinized andnonheparinized plasma, i.e.:

[0077] [{CT (heparinized plasma)-CT(nonheparinizedplasma)}/TC(nonheparinized plasma)×100]

[0078] with CT=clotting time

[0079] The anti-heparin power of the compounds according to theinvention is estimated to be significant when this delta time is lessthan 10%.

[0080] Analysis of Turbidity:

[0081] The turbidity should be as low as possible for an application indiagnosis, and more particularly in a thromboplastin test (PT).

[0082] This turbidity is determined by measuring the OD at 630 nm on aspectrophotometer (Uvikon 940, Kontron).

[0083] Comment: a reagent serving as a negative control, i.e. withoutanti-heparin agent, is of use to measure the influence of thisanti-heparin agent on the turbidity of the reagent.

EXMAPLE 3

[0084] Analysis of the Performance (Anti-Heparin Power and Turbidity) ofVarious Anti-Heparin Agents According to the Invention, Introduced intoa Thromboplastin-Type Reagent (Results Compared to the Control (C)having no Anti-Heparing Agent): Anti-heparin agent at 5 μg/ml Anti-UFHpower Delta time 0/1 IU/ml Turbidity (in %) OD 630 nm 99.2 0.047 C1415-5 PGlu-K₄-(AHA)₂-K₄-AHA-K₄-AHA-K₂ 4.5 0.069 1415-4THC-K₄-(AHA)₂-K₄-AHA-K₄-AHA-K₂ 5.9 0.148 1415-3Hyp-K₄-(AHA)₂-K₄-AHA-K₄-AHA-K₂ 3.4 0.134 1415-2Thi-K₄-(AHA)₂-K₄-AHA-K₄-AHA-K₂ 5.7 0.104 1394Pro-K₄-AHA-K₄-APA-K₃-APA-K₃ 6.4 0.399 1401 Pro-K₄-AHA-K₄-APA-K₄-APA-K₂8.1 0.488 1403P Pro-K₃-AHA-K₄-APA-K₄-APA-K₂ 7.1 0.205 1403PKPro-K₃-AHA-K₄-APA-K₄-APA-K₂ 8.7 0.283 1404 Pro-K₄-AHA-K₃-APA-K₃-APA-K₂7.6 0.147 1405 Pro-K₄-(AHA)₂-K₄-AHA-K₄-AHA-K₂ 4.2 0.323 1408-2Pro-K₄-AHA-K₃-APA-K₄-APA-K₃ 6.9 0.040 1411Pro-K₄-(AHA)₂-K₄-AHA-K₃-AHA-K₂ 6.5 0.086

[0085] with APA corresponding to amino propanoic acid, of formula—(CH₂)₂—C—

[0086] References

[0087] 1. Casu, B. (1985) Adv. Carbohydr. Chem. 43: 51-134

[0088] 2. Constanta, et al. (1993) Angewandte Chemie, Vol. 32, No. 12,1671-1818

[0089] 3. Lormeau, H., et al. (1995) Thromb. Haemostasis 74: 1474-1477

[0090] 4. Mayo, K. M., et al. (1995) Biochem. J. 312: 357-365

[0091] 5. Mardiguian, J. (1992) Ed. Doutremepuich ch. Marcel Dekker inc.7-12

[0092] 6. McKay, D. J., et al. (1986) Eur., J. Biochem. 158: 361-266

[0093] 7. Kurt, F. (1973) Protamines, Institut fur VegetativePhysiologie, Frankfurt, Germany

[0094] 8. Jacques, L. B. (1973) Can. Med. Assoc. J. 108: 1291-1297

[0095] 9. Cundall, R. B., et al. (1979) J. Chem. Soc. Perkins Trans.II:879

[0096] 10. Racanelli, A., et al. (1985) Semin. Thromb. Hemostasis 11:176-189

[0097] 11. Horrow, J. C. (1985) Anesth. Analg. 64: 348-361

[0098] 12. Cook, J. J., et al. (1992) Circulation 85: 1102-1109

[0099] 13. Weller J. M., et al. (1985) J. Allergy. Clin. Immunol. 75:297-303

[0100] 14. Wakefield T. W., et al. (1990) Ann. Surg. 212: 387-395

[0101] 15. Telen A. N., et al. (1977) Thromb. Res. 10, 399-410

[0102] 16. DeLucia, A., et al. (1993) J. Vasc. Surg. 10: 49-58

1. A compound exhibiting anti-heparin activity, of general formula 1:Z-B _(m)-(AXA)_(x)-B_(n)-(AXA)_(y)-B_(o)-(AXA)_(z)-B_(p) in which *Z hasthe general formula:

with Cy=-covalent bond —CH₂— —CO— —CO—S— —(CH₂)_(n)—X— with 1≦n≦2 andX=O—, S—,

with Cx=-covalent bond —(CH₂)_(n)—, 1≦n≦5 or

with R1=—OH, —NH₂ *B represents a basic amino acid; m, n, o and prepresent, independently of one another, an integer of between 2 and 4.*AXA represents a hydrocarbon-based chain corresponding to the formula:—NH—(CH₂)_(n)—CO—, n being an integer of between 2 and 6; x, y and zrepresent, independently of one another, an integer of between 1 and 6.2. A compound as claimed in claim 1, characterized in that Z is selectedfrom the following compounds: 4-aminopyrrolidine-2-carboxylic acidazetidine-2-carboxylic acid 2-carboxymorpholine acid1,3-dihydro-2H-isoindolecarboxylic acid4-hydroxypyrrolidine-2-carboxylic acid indoline-2-carboxylic acidisonipecotic acid pyroglutamic acid pipecolic-2-carboxylic acidpyrrolidine-2-carboxylic acid 2-oxothiazolidine-4-carboxylic acidthiazolidinecarboxylic acid tetrahydroisoquinoline-2-carboxylic acid 3.A compound as claimed in either of claims 1 and 2, characterized in thatZ is pyroglutamic acid (PGlu), of formula:


4. A compound as claimed in one of claims 1 to 3, characterized in thatB is selected from lysine, arginine, ornithine, histidine, homolysineand homoarginine.
 5. A compound as claimed in one of claims 1 to 4,characterized in that B is lysine.
 6. A compound as claimed in one ofclaims 1 to 5, characterized in that m, n and o are equal to 4 and p isequal to
 2. 7. A compound as claimed in one of claims 1 to 6,characterized in that AXA corresponds to the formula NH—(CH₂)₅—CO—(aminohexanoic acid).
 8. A compound as claimed in one of claims 1 to 7,characterized in that x is equal to 2 and y and z are equal to
 1. 9. Acompound as claimed in one of claims 1 to 8, characterized in that itcorresponds to the formula: PGlu-K₄-(AHA)₂-K₄-AHA-K₄-AHA-K₂
 10. Acompound as claimed in one of claims 1 to 9, characterized in that itexhibits an anti-heparin power, determined by the Δ relative clottingtime between a heparinized plasma and a nonheparinized plasma, of lessthan 10%, preferably less than 5% in a clotting test.
 11. A reagent forin vitro diagnosis, comprising a compound as claimed in one of claims 1to
 10. 12. The reagent as claimed in claim 11, for calculating theclotting time of a blood sample, independently of the presence orabsence of heparin in said sample.
 13. The reagent as claimed in claim11, for detecting or assaying various elements of the clotting cascade,independently of the presence or absence of heparin in said sample. 14.The reagent as claimed in claim 11, characterized in that it is a PTreagent.
 15. The reagent as claimed in claim 11, characterized in thatit comprises the compound of formula: PGlu-K₄-(AHA)₂-K₄-AHA-K₄-AHA-K₂16. The reagent as claimed in claim 11, characterized in that itexhibits a turbidity, measured by an OD at 630 nm, of less than 0.5,preferably less than 0.1.
 17. The reagent as claimed in claim 11,comprising a compound of general formula 1 at a final concentration ofbetween 2 and 10 μg/ml.
 18. The reagent as claimed in claim 11,characterized in that the final concentration of the compound in saidreagent is 5 μg/ml.
 19. The use of a compound as claimed in one ofclaims 1 to 10, in a reagent for in vitro diagnosis, for calculating theclotting time of a blood sample, or detecting or assaying in a bloodsample various elements of the clotting cascade, independently of thepresence or absence of heparin in said sample.
 20. The use of a compoundas claimed in one of claims 1 to 10, as an anti-heparin agent forpreparing a medicinal product.